Samples were embedded in paraffin at a thickness of 4 nm. Deparaffinization and rehydration were performed on each slide. To eliminate aldehyde linkages from antigens, they were re-extracted using a pressure cooker and 0.01 M citrate buffer (pH 6). The slides were incubated with HSP90B1 antibody (1: 2000; ab238126, Abcam) overnight. After incubating the HRP-labelled secondary antibody for 1 h, immunodetection was performed the following day using diaminobenzidine following the manufacturer’s instructions (Yuan et al., 2021 (link)). Two independent pathologists, each of whom was blinded to the other’s clinical data, evaluated the HSP90B1 staining. A semi-quantitative immune response score (IRS) system incorporating distribution regions and staining intensities was used for the HSP90B1 staining procedure. The immunological staining intensity score ranged from 0 to 3 (0, no reaction; 1, weak response; 2, moderate response; 3, robust response). The proportions were separated into 1 (0%–25%), 2 (26%–50%), 3 (51%–75%) and 4 (76%–100%) (Zhu et al., 2020 (link)). The final score was obtained by adding the intensity score and the proportional score. The results were as follows: 0–5, low; 6–7, high.
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